Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Patent
1997-02-18
1999-08-10
Beisner, William H.
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
4352887, 4352865, 4352879, C12M 140
Patent
active
059358467
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The invention discloses a process and a device for determining the activity of enzymes in liquids, or the concentration and/or activity of inhibitors in liquids.
The determination of enzyme activities in extracts of plants, suspensions of bacteria, homogenates and body fluids such as blood serum, blood plasma, urine, punctate, liquor, cell lines or homogenated tissue has obtained an essential importance for diagnosis, follow up and therapy control.
For example, the discrimination between enzyme proteins and the other proteins in blood serum by chemical means is exceedingly questionable, because the concentrations of the individual enzymes in body fluids are extremely small. The concentration of glutamate-oxaloacetate transaminase in blood serum of a healthy individual is 0,1 .mu.g/ml, for example. As for comparison, the total protein concentration in blood serum is in the range between 60 to 80 mg/ml, that is, the ratio of these two concentrations is about 1:700.000. Since the determination of the enzyme concentration by chemical means is questionable, the activity of the enzyme is calculated from the rate of its reaction with a suitable substrate.
The so called ELISA assay is known from clinical chemistry. This immunoassay measures the concentration of an enzyme and its corresponding enzym-inhibitor complex present in a tissue or any other sample. Measuring the enzyme activity by this method is impossible, as it measures concentrations regardless to the actual status of the enzyme, active or inhibited.
According to known techniques the activity of an enzyme is measured in such cases by removing at first the enzyme inhibitors corresponding to the enzyme and afterwards determining the activity of the enzyme. The known processes are very laborious, referring to the samples to be measured being tissues as well as to the method, how to remove the enzyme inhibitors from the sample. This method comprises adding to the sample a substance capable of binding the enzyme inhibitors, incubating the so manipulated sample during a definite time to complete the binding of the enzyme inhibitors along with as much homogenous mixing as possible of the sample with the said substance and finally, separating the so manipulated sample from the said substance.
SUMMARY OF THE INVENTION
Starting from this point, the purpose of the present invention is to disclose a process and a device for measuring the activity of enzymes in fluids by running the method of measurement largely or completely automatically and also effectively, and for determining the concentration and/or activity of inhibitors in fluids additionally or alternatively.
The inventive process manages the above mentioned problem according to the features of the claims 1 or 23. Accordingly, a process is arranged for measuring enzyme activities in fluids, which comprises withdrawing enzyme inhibitors, that correspond to at least one of the enzymes in the sample, or enzymes, that correspond at least to one inhibitor, adding a substrate to the sample manipulated in this manner, so as to get cleavage products from the substrate by reacting with the enzyme, and detecting the increasing concentration per unit of time of at least one of these cleavage products during an incubation time. The said process is characterized by withdrawing the enzyme inhibitors or enzymes from the sample by means of chromatography.
According to the said invention, it was discovered, that it is possible to remove the enzyme inhibitors from the sample without as much homogenous mixing as possible of the corresponding substance and the sample, and without a following laborious separation process. In connection with this, it was discovered, that one can perform mixing and separation virtually in one step. The said invention has the special advantage, that it enables one now to measure enzyme activities in fluid samples, that is, all kinds of body fluids as well as homogenated tissues, whereby the withdrawing of the enzyme inhibitors by means of chromatography makes it pos
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Anastasi et al., Cystatin, a protein inhibitor of cysteine proteinases, Biochem. J., 211: 219-138, Dec. 13, 1982.
Koohmaraie et al., Comparisons of Four Methods for Quantification of Lysosomal Cysteine Proteinase Activities, J. Anim. Sc. 68: 2362-2370, 1990 .
Schumacher Johannes
Werle Bernd
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